RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 from Inhibition of Reverse Transcription and Acceleration of Uncoating

Rhesus TRIM5 alpha(TRIM5 alpha(rh)) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5 alpha(rh) to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5 alpha to block reverse transcription, we studied a set of TRIM5 alpha(rh) RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5 alpha(rh) RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5 alpha. Similar effects of this particular group of mutations were observed with human TRIM5 alpha inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5 alpha(rh) RING domain variants also prevented the degradation of TRIM5 alpha(rh) that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5 alpha to accelerate uncoating. Collectively, these results suggest that TRIM5 alpha(rh) blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.